West Nile virus (WNV) is a mosquito-borne virus that has been introduced to the U.S. in 1999 (Solomon et al, 1998; Gubler, 1998 Monath, 2005, Tomorri, 1999). Since the initial outbreak, there have been cases reported in all but two states in the U.S. WNV has increasingly become a public health threat, causing hundreds of deaths and tens of thousands of infections (Solomon et al, 1998; Gubler, 1998 Monath, 2005, Tomorri, 1999). Although there has been progress in vaccine development to prevent WNV encephalitis in humans (Rossi 2005), there is still no effective vaccine or antiviral drug therapy (Rossi 2005). Currently, the only available treatment is supportive, and the only existing means of prevention is mosquito control, which is also of limited success. It is also considered to be an agent of bioterrorism concern (Monath 2005), and therefore, safe and effective antiviral drugs to treat WNV infection are urgently needed.
WNV is a positive, single stranded RNA virus (Rossi 2005). It belongs to the Flaviviridae family, and Flavivirus genus. Many flaviviruses are significant human pathogens. In addition to WNV, this flavivirus sero-complex includes Japanese encephalitis virus (JEV), St. Louis encephalitis (SLEV), Alfuy virus (AV), Koutango virus (KV), Kunjin virus (JV), Cacipacore virus (CV), Yaounde virus (YV), and Murray Valley encephalitis virus (MVEV). The Flaviviridae family also includes the Tick-borne encephalitis virus (TBEV), Dengue virus (including the four serotypes of: DENV-1, DENV-2, DENV-3, and DENV-4), and the family prototype, Yellow Fever virus (YFV).
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality. A combined toll of hundreds of millions of infections around the world annually coupled with the lack of sustained mosquito control measures, has distributed flaviviruses throughout the tropics, subtropics, and temperate areas. As a result, over half the world's population is at risk for flaviviral infection. Further, modern jet travel and human migration have raised the potential for global spread of these pathogens. Strains of WNV are categorized into two different phylogenetic lineages, namely, lineage I and II, which share 75% nucleotide sequence identity (Lanciotti, R et al, (2002) Virology 298:96-105). Lineage I strains have been isolated from human and equine epidemic outbreaks from around the world and constitute the main form of human pathogen. Sequence analysis indicates that the current epidemic strain in North America belongs to lineage I. Lineage II strains are rarely isolated from humans and are geographically restricted primarily to sub-Saharan Africa and Madagascar. The differences in disease patterns of lineage I and II strains are postulated to be the result of differences in vector competence (host compatibility), virulence, and transmission cycles of the strains, as well as, host immunity (Beasley, D. W. C. et al, (2001) International Conference on the West Nile Virus, New York Academy of Science Poster Section 1:5). Sequence analysis showed that the strain in North America is closely related to other human epidemic strains isolated from Israel, Romania, Russia, and France, all of which belong to lineage I (Lanciotti, R. et al. (1999) Science 286:2333-2337).
The flavivirus genome, including the genome of WNV, is a single positive-sense RNA of approximately 10,500 nucleotides containing short 5′ and 3′ untranslated regions (UTR), a single long open reading frame (ORF), a 5′ cap region, and a non-polyadenylated 3′ terminus. The entire genome is transcribed as a single polycistronic messenger RNA molecule, which is then translated as a polyprotein. Individual proteins are subsequently produced by proteolytic processing of the polyprotein, which is directed by viral and host cell proteases (Chambers, T. J. et al, (1990) Ann. Rev. Microbiol. 44: 649-688; Lindenbach, B. D. and C. M. Rice, (2001) In D. M. Knipe and P. M. Howley (ed), Fields virology, 4.sup.th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.).
During the replication cycle of flaviviruses, especially WNV, synthesis of positive and negative (hereafter referred to as plus (+) and minus (−), respectively) sense RNAs is asymmetric. In the case of WNV, plus-sense RNAs are produced in 10- to 100-fold excess over minus-sense RNA. Regulatory sequences in the 3′ UTR are believed to function as a promoter for initiation of minus-strand RNA synthesis. Deletion of this region ablates viral infectivity (Brinton, M. A. et al, (1986) Virology 162: 290-299; Proutski, V., et al (1997) Nucleic Acids Res. 25: 1194-1202; Rauscher, S., et al (1997) RNA 3: 779-791).
WVN genome is 12 kilobases in length and has a 5′ and 3′ non-translated region (NTR). The coding sequences specify a single polyprotein, which is proteolytically processed into approximately a dozen functional proteins by both viral and cellular proteases (5). The genes for structural proteins, namely capsid (C), membrane (M; which exists in cells as its precursor, prM), and envelope (E) are located in the 5′ region of the genome, where those for the nonstructural proteins (NS), namely, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (5) are located at the 3′ portion of the genome. WNV can infect many cell types and produce cytopathic plaques. However, due to the highly infectious nature, infections must be carried out in BL3 labs, limiting the use of the plaque assay to screen large number of compounds.
The development of therapeutic drugs and/or vaccines to treat and/or immunize against WNV and other flavivirus infections is urgently needed and of great importance to global public health. To achieve this goal, high-throughput screening assays were developed to facilitate the identification of novel chemotherapeutics effective against flaviviruses or vaccines capable of establishing a protective immune response to flaviviruses (see U.S. Patent Application Publication No. 2005/0058987 to Shi et al.). Two general strategies to be adapted for the screening and identification of novel chemotherapeutic antiflaviviral compounds and/or vaccines are based on biochemical and genetic approaches.
Assays for screening antiviral compounds that are based on biochemical approaches typically involve testing compounds for activities that limit or inhibit viral enzymes or proteins that are essential for viral propagation. For example, NS3, which has protease, helicase and NTPase activity, and NS5, which has an RNA-dependent RNA polymerase and methyltransferase activity, are key components of viral replication complex and thus, are ideal targets for antiviral screening. Further, three-dimensional structures of viral proteins, if available, can afford the possibility for rational design of drugs that will inhibit their activity, i.e., designing drugs based on the knowledge of the structure and shape of the active sites of the protein. For example, the crystal structures of the DENV NS3 protease domain and NS5 cap methyltransferase fragment have been solved and thus, the possibility of rationally designing small molecules to inhibit the active sites of NS3 and NS5 is feasible. Although biochemical approaches are capable of identifying potential viral inhibitors, they are limited in their overall efficiency since only a single enzyme or protein can be tested for any potential assay. Thus, individual assays would be required to screen for inhibitors of each given viral target protein.
In contrast, assays utilizing a genetic approach, which are usually cell-based, offer a number of advantages over biochemical approaches. One major advantage of a genetic approach based assay is that multiple viral protein targets can be analyzed simultaneously. A second major advantage is that, since genetic assays involve the use of living cells and the uptake of compounds therein, the screening assay is administered in a more authentic therapeutic environment. Accordingly, inhibitors identified through cell-based assays typically have a higher success rate in subsequent animal experiments.
A cell-based assay available for screening for flaviviral inhibitors involves the infection of cultured cells with virus and the subsequent monitoring for potential inhibition in the presence of a potential inhibitor through observation or quantification of cytopathic effects (J. D. Morrey et al., Antiviral Res (2002) 55:107-116; I. Jordan, J. Infect. Dis. (2000) 182:1214-1217) or quantification of viral RNA by reverse transcriptase (RT)-PCR(S. F. Wu, J. Virol. (2002) 76:3596-3604). These assays are highly labor-intensive and impossible to use when screening compound libraries in large quantities.
Genetic high-throughput cell-based screening assays for the rapid screening and identification of potential inhibitors from compound libraries utilizing cDNA clones of RNA viruses are preferred screening tools for identifying potential inhibitors.
For example, two kinds of reverse genetics systems, full-length infectious cDNA clones and replicons, have been developed for a number of flaviviruses (A. A. Khromykh, et al., J. Virol. (1997) 71:1497-1505; M. S. Campbell, et al., Virol. (2000) 269:225-237; R. J. Hurrelbrink, et al., J. Gen. Virol. (1999) 80:3115-3125; M. Kapoor, et al., Gene (1995) 162:175-180; A. A. Khromykh et al., J. Virol. (1994) 68:4580-4588; C. J. Lai et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:5139-5143; C. W. Mandl et al., J. Gen. Virol. (1997) 78:1049-1057; C. M. Rice et al., Science (1985) 229:726-733; H. Sumiyoshi et al., J. Virol. (1992) 66:5425-5431; S. Polo et. al., J. Virol. (1997) 71:5366-5374), including lineage II WNV (V. F. Yamshchikov et al., Virology (2001) 281:294-304). Reporter genes can be engineered into the reverse genetics systems to allow for the monitoring of viral replication levels in the presence of potential inhibitors.
U.S. Patent Application Publication No. 2005/0058987 to Shi et al. describes high-throughput cell-based assays for the rapid screening and identification of potential inhibitors from compound libraries utilizing a reverse genetics system developed for lineage I WNV cDNA clone and lineage I WNV replicon.
WNV subgenomic RNAs capable of replicating within cells (replicons) have been reported. (Rossi 2005; Guo 2005). The replicon RNA genome typically contains the 5′ Nontranslated region (5′ NTR), a portion of the Core coding region, a polyprotein encoding NS 1 through NS5, and the 3′ NTR. Like replication of the WNV genome (reviewed in reference (Monath 2005)), in the replicon cells, the viral RNA dependent RNA polymerase, NS5B, in conjunction with other viral nonstructural proteins and possibly cell factors (Monath 2005), synthesize a minus strand RNA from the replicon subgenomic RNA template. The minus strand RNA in turn serves as templates for the synthesis of new genomic and message RNAs. Although data from studies of Kunjin virus suggest that both plus and minus strand RNA synthesis can occur in the absence of protein synthesis once the replication cycle establishes, viral protein synthesis is a prerequisite for replication of nascent RNAs (Guo 2005). In addition to a selectable marker, neomycin phosphotransferaser gene, which is used for selection of the stable cell line, a luciferase reporter gene was also inserted into the RNA with its translation driven by the EMCV IRES (Guo 2005, Monath 2005). The expression of the reporter gene depends on the replication of the replicon RNA and can be easily monitored for identification of antiviral compounds (Guo 2005).
For the purposes of drug screening it is preferable to use human epidemic-causing lineage I strains for assay setup to ensure that the identified compounds have a direct relevance to human disease.
Effective chemotherapeutics to treat WNV and other flaviviruses, known and emerging, are urgently needed. Although a limited number of inhibitors of flaviviruses have been identified, many of these have severe side effects, are not specific to flaviviruses, and are not known to be clinically effective and/or useful. For example, recent evidence suggested the use of nucleoside analogs as potential inhibitors of flaviviruses. Specific examples include inhibitors of orotidine monophosphate decarboxylase, inosine monophosphate dehydrogenase, and CTP synthetase. Although it appeared that these inhibitors may have been effective in virus infected Vero cells, their effectiveness in humans or animals (i.e., in vivo) is not known. Additionally, as these nucleoside analogs are broad-spectrum inhibitors of purine and pyrimidine biosynthesis, the occurrence of side effects and lack of flaviviral specificity would further limit their usefulness in a clinical setting.
Hepatitis B Virus (HBV) is a causative agent of acute and chronic liver disease including liver fibrosis, cirrhosis, inflammatory liver disease, and hepatic cancer that can lead to death in some patients. Although effective vaccines are available, there are still more than 300 million people worldwide, i.e., 5% of the world's population, chronically infected with the virus (Locamini, S. A., et. al., Antiviral Chemistry & Chemotherapy (1996) 7(2):53-64). Such vaccines have no therapeutic value for those already infected with the virus. In Europe and North America, between 0.1% to 1% of the population is infected. Estimates are that 15% to 20% of individuals who acquire the infection develop cirrhosis or another chronic disability from HBV infection. Once liver cirrhosis is established, morbidity and mortality are substantial, with about a 5-year patient survival period (Blume, H., E., et. al., Advanced Drug Delivery Reviews (1995) 17:321-331). It is therefore necessary and of high priority to find improved and effective anti-HBV anti-hepatitis therapies.
Other hepatitis viruses significant as agents of human disease include Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis Delta, Hepatitis E, Hepatitis F, and Hepatitis G (Coates, J. A. V., et. al., Exp. Opin. Ther. Patents (1995) 5(8):747-756). In addition, there are animal hepatitis viruses that are species-specific. These include, for example, those infecting ducks, woodchucks, and mice.
WO 2001/10429A2 to Dwek et al. describes methods of inhibiting morphogenesis of a pestivirus or flavivirus and treatment of hepatitis B and C infection by administration of a long chain N-alkyl amino or imino compound or an oxa-substituted derivative thereof including N-(7-oxa-nonyl)-1,5-dideoxy-1,5-imino-D-galactitol.
US 2006/0074107A1 to Butters et al. describes an agent capable of increasing the rate of neuronal glycolipid degradation for the treatment of mucopolysaccharide diseases having the following formula
wherein R is C1-16 straight or branched-chain alkyl, optionally substituted by C3-7 cycloalkyl, and optionally interrupted by —O—, the oxygen being separated from the ring nitrogen by at least two carbon atoms, or C1-10 alkylaryl where aryl is phenyl, pyridyl, thienyl or furyl wherein phenyl is optionally substituted by one or more substituents selected from F, Cl, Br, CF3, OCF3, OR1 and C1-6 straight or branched-chain alkyl; and R1 is hydrogen, or C1-6 straight or branched-chain alkyl.
U.S. Pat. No. 6,809,083 to Mueller et al. described the use of N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds for treating hepatitis virus infections. However, Mueller et al. did not describe using N-substituted alkylhydroxylcycloalkyl derivatives.
1,5-dideoxy-1,5-imino-D-glucitol (also known as 1-deoxynojirimycin, DNJ) and its N-alkyl derivatives (together, “imino sugars”) are known inhibitors of the N-linked oligosaccharide processing enzymes alpha glucosidase I and II (Saunier et al., J. Biol. Chem. (1982) 257:14155-14161 (1982); Elbein, Ann. Rev. Biochem. (1987) 56:497-534). As glucose analogs, they also have potential to inhibit glucose transport, glucosyl-transferases, and/or glycolipid synthesis (Newbrun et al., Arch. Oral Biol. (1983) 28: 516-536; Wang et al., Tetrahedron Lett. (1993) 34:403-406). Their inhibitory activity against glucosidases has led to the development of these compounds as anti-hyperglycemic agents and antiviral agents. See, for example, PCT International Publication WO 87/03903 and U.S. Pat. Nos. 4,065,562; 4,182,767; 4,533,668; 4,639,436; 4,849,430; 4,957,926; 5,011,829; and 5,030,638.
Glucosidase inhibitors such as N-alkyl-1,5-dideoxy-1,5-imino-D-glucitol compounds wherein the alkyl group contains between three and six carbon atoms have been shown to be effective in the treatment of Hepatitis B infection (PCT International Publication WO 95/19172). For example, N-(n-butyl)deoxynojirimycin (N-butyl-DNJ; N-(n-butyl)-1-5-dideoxy-1,5-imino-D-glucitol) is effective for this purpose (Block, T. M., Proc. Natl. Acad. Sci. USA (1994) 91:2235-2239; Ganem, B. Chemtracts: Organic Chemistry (1994) 7(2), 106-107). N-butyl-DNJ has also been tested as an anti-HIV-1 agent in HIV infected patients.
The deoxynojirimycins (DNJs) are iminocyclitol glucomimetics that inhibit ER glucosidases by competing with glucose (Tan et al., 1991). Although millimolar amounts of DNJ are needed to achieve substantial inhibition of the endoplasmic reticulum (ER) enzyme in tissue culture and to achieve 50% inhibition of virus yields against sensitive viruses, the drug is well tolerated with little apparent cytotoxicity. Modification of DNJs by adding side chains to the ring nitrogen atom has been shown to affect their activity and toxicity. For example, N-nonyl-DNJ (NNDNJ), a nine carbon alkyl side chain derivative of DNJ (FIG. 1) was shown to be an effective antiviral agent against HBV (Block et al., 1994) and BVDV (Durantel et al., 2001; Jordan et al., 2002; Zitzmann et al., 1999). However, NNDNJ possessed more toxic activity (Mehta et al., 2001; Zitzmann et al., 1999) and had a selectivity index that was deemed to be too narrow for clinical development. Therefore, compounds with similar efficacy but better toxicity profiles were sought.
Despite the current developments, there is a need in the art for novel flavivirus replication inhibitors, particularly, non-nucleoside based compounds, which have better toxicity profiles.
All references cited herein are incorporated herein by reference in their entireties.